The development of methodologies to produce nanoparticles with bio-responsive properties has opened the way for producing useful tools for molecular diagnostics, therapeutics and biotechnology [1]. Metal, semiconductor and magnetic colloidal nanoparticles are presently under intensive study for potential applications [2].
Nanoparticles containing paramagnetic materials such as iron oxide have been made which exhibit unusually strong magnetic properties under external magnetic fields. These magnetic nanoparticles can be used in many biomedical applications, including cell separation, in vivo cell and tissue labelling, contrast enhancement in magnetic resonance imaging, tumour targeting, hyperthermia therapies and drug delivery.
For such applications, the nanoparticles should preferably be small enough to avoid provoking an immune response and to be taken up by cells, where necessary. It is also useful if the size of the particles can be controlled as the particles should be of approximately the same size so they display the same magnetic properties. The particles should also preferably be chemically stable, so they are not broken down by the body.
In is also preferred that magnetic nanoparticles for use in biomedicine are soluble, especially in water, in order that they may be stored and administered effectively. Ideally, such particles would be stable in solution and would not aggregate, either when stored before use or in the body. Magnetic nanoparticles tend to clump together in solution because they attract each other. If this happened in the body it could impede blood flow and potentially be dangerous; in colloidal solution it would make the colloid difficult to use.
Previously, commercially available iron oxide particles have been used in cell sorting and separation [3]. Monodisperse magnetic nanoparticles of Fe/Pt [4], Co and Co/Fe [5], Fe [6], and iron oxides [7] have recently been synthesised by solution chemistry for materials applications.[8]. Iron oxide nanoparticles coated with cross-linked dextran to prevent clumping have also been described, see for example WO 03/005029.
Ideally, the magnetic nanoparticles are made of elemental magnetic metal rather than metal oxide, as elemental metal is a better enhancer of magnetic imaging. However, such nanoparticles are often chemically unstable, as the metal may oxidise. One possibility for increasing the chemical stability of magnetic nanoparticles is to synthesise them from a magnetic metal with a passive metal to stabilise the magnetic metal.
US 2002/0068187 discloses surfactant protected gold-iron core-shell nanoparticles synthesised by means of reverse micelles. However, this method is complex, requiring three synthesis steps. The multi-layered composition of the resulting particles also increases the lower size limit for the particles, which can be a disadvantage if very small particles are required [14].
U.S. Pat. No. 6,254,662 discloses use of FePt and CoPt alloy nanoparticles to form nanocrystalline thin films on a solid surface, for use in making ultra-high density recording media. Other uses of the films are mentioned in the parent, including use as magnetic bias films and magnetic tips for magnetic force microscopy, but biomedical applications are not envisaged.
For many of the applications described above, it is necessary to link the nanoparticles to biologically active molecules such as ligands that bind to intracellular or extracellular molecules. Such ligands may for example be carbohydrate, nucleic acid or protein.
U.S. Pat. No. 6,514,481 discloses iron oxide nanoparticles coated with a silica shell, where the shell is linked to a targeting molecule such as a peptide via a spacer molecule. WO 02/098364 and WO 01/19405 disclose magnetic metal oxide nanoparticles coated with dextran and functionalised with peptides and oligonucleotides. Similar strategies have been used to prepare nanoparticles for intracellular labelling [9] and as nanosensors.[10]. All these methods are time-consuming multi-step methods requiring that the nanoparticles be coated with dextran or silica, the coated nanoparticles be functionalised so they will bind the ligand, and finally that the ligand be bound to the nanoparticles.
WO 03/073444 discloses superparamagnetic nanoparticles having a cores formed from Au and Fe metal atoms in a ratio of at least 3:7. The application says that ligands can be linked to the core via a sulphide group and that the nanoparticles are used for forming nanoelectronic devices. The cores of the nanoparticles have diameters in the range of 5 nm to 50 nm.
WO 02/093140 discloses magnetic nanowires which comprise one or more segments and functional groups or ligands associated with a at least one of said segments. The nanowires have a diameter in the range of about 10-300 nm and a length from 10 nm to tens of microns. The segments of the nanowires may be formed from materials such as gold, silver, platinum, copper, iron and cobalt in pure or alloyed form and the functional groups may be atoms or groups of atoms that are capable of further chemical reactivity such as reacting with a ligand to attach the ligand to the wire, or to bind a target molecule. Although a range of possible ways of associating the ligands and the nanowires are proposed, the examples rely on the ionic interaction between ligands containing carboxylic acid groups and the nanowire.
U.S. Pat. No. 6,531,304 discloses a nanoscale colloid formed from metal alloys which is reacted and non-covalently binds a polysaccharide or sugar “modifier”.
WO 02/32404 discloses water soluble nano-tools for studying carbohydrate mediated interactions [11], [12]. These tools are gold glyconanoparticles and cadmium sulphide glyco-nanodots incorporating carbohydrate antigens. These water soluble gold and semiconductor nanodots are stable for months in physiological solutions and present exceptionally small core sizes. They are resistant to glycosidases and do not present cytotoxicity. They are also useful platforms for basic studies of carbohydrate interactions [13] and are tools for biotechnological and biomedical applications. However, these nanoparticles are not magnetic.
There is therefore a continuing need in the art for stable magnetic nanoparticles which are bound to ligands to make them suitable for biomedical uses, which can be synthesised to a desired size, and which can be produced by a simple, reliable synthesis method.